Nongenomic testosterone calcium signaling. Genotropic actions in androgen receptor-free macrophages

Steroid hormones exert genotropic actions through members of the nuclear receptor family. Here, we have demonstrated genotropic actions of testosterone that are independent of intracellular androgen receptors (iAR). Through plasma membrane androgen receptors (mAR), testosterone induces a rapid rise in the intracellular free Ca(2+) concentration of iAR-free murine RAW 264.7 macrophages. This nongenomic testosterone signaling, which is independent of both iAR and estrogen receptors, does not in itself activate either the mitogen-activated protein kinase (MAPK) families ERK1/2, p38, and JNK/SAPK, the stably and transiently transfected c-fos promoter, or NO production. In the context of lipopolysaccharide (LPS) signaling, however, testosterone attenuates LPS activation of the c-fos promoter and NO production, which is abolished by the intracellular Ca(2+) chelator BAPTA. Testosterone also attenuates the LPS activation of p38 but not that of ERK1/2 and JNK/SAPK, and this attenuation is abrogated by BAPTA. Moreover, the p38 inhibitor, SB 203580, largely reduces LPS activation of the c-fos promoter and NO production, and the remaining levels are no longer regulated by testosterone. This study is the first to provide information on genotropic actions of mAR-mediated nongenomic testosterone Ca(2+) signaling by cross-talk with the LPS signaling pathway through p38 MAPK with impact on cell function.     

Centromere structure and function analysis in wheat–rye translocation lines

1RS.1BL translocations are centric translocations formed by misdivision and have been used extensively in wheat breeding. However, the role that the centromere plays in the formation of 1RS.1BL translocations is still unclear. Fluorescence in situ hybridization (FISH) was applied to detect the fine structures of the centromeres in 130 1RS.1BL translocation cultivars. Immuno‐FISH, chromatin immunoprecipitation (ChIP)‐qPCR and RT‐PCR were used to investigate the functions of the hybrid centromeres in 1RS.1BL translocations. New 1R translocations with different centromere structures were created by misdivision and pollen irradiation to elucidate the role that the centromere plays in the formation of 1RS.1BL translocations. We found that all of the 1RS.1BL translocations detected contained hybrid centromeres and that wheat‐derived CENH3 bound to both the wheat and rye centromeres in the 1RS.1BL translocation chromosomes. Moreover, a rye centromere‐specific retrotransposon was actively transcribed in 1RS.1BL translocations. The frequencies of new 1RS hybrid centromere translocations and group‐1 chromosome translocations were higher during 1R misdivision. Our study demonstrates the hybrid nature of the centromere in 1RS.1BL translocations. New 1R translocations with different centromere structures were created to help understand the fusion centromere used for wheat breeding and for use as breeding material for the improvement of wheat.     

Hypoxic preconditioning‐induced autophagy enhances survival of engrafted endothelial progenitor cells in ischaemic limb

Recent clinical studies have suggested that endothelial progenitor cells (EPCs) transplantation provides a modest benefit for treatment of the ischaemic diseases such as limb ischaemia. However, cell‐based therapies have been limited by poor survival of the engrafted cells. This investigation was designed to establish optimal hypoxia preconditioning and evaluate effects of hypoxic preconditioning‐induced autophagy on survival of the engrafted EPCs. Autophagy of CD34⁺VEGFR‐2⁺ EPCs isolated from rat bone marrow increased after treatment with 1% O₂. The number of the apoptotic cells in the hypoxic cells increased significantly after autophagy was inhibited with 3‐methyladenine. According to balance of autophagy and apoptosis, treatment with 1% O₂ for 2 hrs was determined as optimal preconditioning for EPC transplantation. To examine survival of the hypoxic cells, the cells were implanted into the ischaemic pouch of the abdominal wall in rats. The number of the survived cells was greater in the hypoxic group. After the cells loaded with fibrin were transplanted with intramuscular injection, blood perfusion, arteriogenesis and angiogenesis in the ischaemic hindlimb were analysed with laser Doppler‐based perfusion measurement, angiogram and the density of the microvessels in histological sections, respectively. Repair of the ischaemic tissue was improved significantly in the hypoxic preconditioning group. Loading the cells with fibrin has cytoprotective effect on survival of the engrafted cells. These results suggest that activation of autophagy with hypoxic preconditioning is an optimizing strategy for EPC therapy of limb ischaemia.     

Polydatin alleviated radiation‐induced lung injury through activation of Sirt3 and inhibition of epithelial–mesenchymal transition

Radiation‐induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy. It is characterized with two main features including early radiation pneumonitis and fibrosis in later phase. This study was to investigate the potential radioprotective effects of polydatin (PD), which was shown to exert anti‐inflammation and anti‐oxidative capacities in other diseases. In this study, we demonstrated that PD‐mitigated acute inflammation and late fibrosis caused by irradiation. PD treatment inhibited TGF‐β1‐Smad3 signalling pathway and epithelial–mesenchymal transition. Moreover, radiation‐induced imbalance of Th1/Th2 was also alleviated by PD treatment. Besides its free radical scavenging capacity, PD induced a huge increase of Sirt3 in culture cells and lung tissues. The level of Nrf2 and PGC1α in lung tissues was also elevated. In conclusion, our data showed that PD attenuated radiation‐induced lung injury through inhibiting epithelial–mesenchymal transition and increased the expression of Sirt3, suggesting PD as a novel potential radioprotector for RILI.     

Brefeldin A enhances docetaxel-induced growth inhibition and apoptosis in prostate cancer cells in monolayer and 3D cultures

Docetaxel is a commonly used chemotherapeutic drug for patients with late stage prostate cancer. However, serious side effect and drug resistance limit its clinical success. Brefeldin A is a 16-membered macrolide antibiotic from mangrove-derived Fungus Aspergillus sp. (9Hu), which exhibited potent cytotoxicity against human cancer cells. In the present study, we determined the effect of brefeldin A on docetaxel-induced growth inhibition and apoptosis in human prostate cancer PC-3 cells. Brefeldin A in combination with docetaxel inhibited the growth of PC-3 cells in monolayer and in three dimensional cultures. The combination also potently stimulated apoptosis in PC-3 cells as determined by propidium iodide staining and morphological assessment. Mechanistic studies showed that growth inhibition and apoptosis in PC-3 cells treated with brefeldin A and docetaxel were associated with decrease in the level of Bcl-2. The present study indicates that combined brefeldin A with docetaxel may represent a novel approach for improving the efficacy of docetaxel, and Bcl-2 may serve as a target for brefeldin A to enhance the effects of docetaxel chemotherapy.     

Design,synthesis and biological activity evaluation of novel 2,6-difluorobenzamide derivatives through FtsZ inhibition

Novel series of 3-substituted 2,6-difluorobenzamide derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their in vitro antibacterial activity against various phenotype of Gram-positive and Gram-negative bacteria, and their cell division inhibitory activity against three representative strains. As a result, 3-chloroalkoxy derivative 7, 3-bromoalkoxy derivative 12 and 3-alkyloxy derivative 17 were found to exhibit the best antibacterial activity against Bacillus subtilis with MICs of 0.25–1 μg/mL, and good activity (MIC < 10 μg/mL) against both susceptible and resistant Staphylococcus aureus. Additionally, all the three compounds displayed potent cell division inhibitory activity with MIC values of below 1 μg/mL against Bacillus subtilis and Staphylococcus aureus.     

染色质调节元件与不同启动子的相互作用对基因表达调控的影响

为探究DNA序列元件对不同启动子调节转基因稳定表达的影响,利用遍在染色质开放元件 (Ubiquitous chromatin opening elements,UCOE) 和基质黏附序列 (Scaffold/matrix-attachment regions,MAR) 分别与含增强子的oct4基因启动子、含CpG岛的sox2基因启动子和不含调控元件的nanog基因启动子以及同时包含增强子和CpG岛的CMV启动子组合构建pOCT4-MAR、pOCT4-UCOE、pSOX2-MAR、pSOX2-UCOE、pNANOG-MAR、pNANOG-UCOE、pCMV-UCOE、pCMV-MAR等质粒,分析这些质粒稳定转染后的表达量和嵌合表达差异。结果发现,UCOE与含增强子元件的oct4启动子组合能较稳定高效表达,而MAR与含CpG岛的sox2启动子组合能较稳定高效表达。利用排除位置效应原因的嵌合表达对染色质高级结构调控基因表达的稳定性分析表明：(1) 通常情况下UCOE比MAR调节的表达载体的表达更高效和更稳定;UCOE连接含CpG岛的启动子形成开放染色质调节的高表达更稳定;(2) MAR与启动子上TATA盒或增强子可能通过染色质环产生高表达,但相对不稳定。结论：染色质调节元件UCOE和MAR与启动子调控元件之间能通过染色质开放状态或染色质环调控基因稳定表达。     

应用iTRAQ技术分析滩羊二毛期不同花穗型裘皮差异蛋白

为了解二毛期时滩羊串子花型裘皮与其它类型裘皮中蛋白质的差异,本试验采用iTRAQ技术及LC-MS/MS蛋白质组学研究方法,对串子花型、软大花型、绿豆丝型及其它不规则型花穗裘皮蛋白质进行鉴定和筛选,并运用Proteome Discoverer l.4软件进行定量分析,结合数据库搜索,鉴定出具有显著表达差异的蛋白,同时应用生物学技术对其进行GO和Pathway分析。结果显示4类花穗型裘皮共检测出2 886个蛋白,其中有135个、142个、113个差异蛋白分别存在于软大花型与串子花型、绿豆丝型与串子花型、其它不规则型与串子花型3个对比组中。对有表达差异的蛋白进行分析,发现膜联蛋白与血管内皮生长因子可能与软大花型毛股形成相关,KAP3和KAP6可能与滩羊串子花型毛股结构相关。研究发现滩羊不同二毛裘皮蛋白水平上的差异,可为选育优良的滩羊串子花型二毛裘皮提供理论基础。